Commercial antibodies are commonly formulated in phosphate buffer. Also TNFR:Fc is commonly buffered in sodium phosphate (EP1478394, WO 03/072060 A2). Currently, e.g. the TNFR:Fc protein Etanercept is marketed under the tradename Enbrel® having a composition as shown in Table 1.
TABLE 1Composition of Etanercept (Enbrel®)[mg/mL][mM]Etanercept500.3Sucrose1029Sodium chloride5.8100L-arginine hydrochloride5.325Sodium phosphate2.619monobasicSodium phosphate dibasic0.96WFIad 0.5/1.0 mlpH6.3 ± 0.2
Aggregation of antibody products can be controlled by the addition of small amphiphilic molecules. Thereof, L-arginine is the amino acid of choice in suppressing protein interactions in commercial formulations (Baynes et al (2005) 44(12):4919-25; EP1478394). Being a polar additive, it prevents the aggregation of protein folding intermediates.
Like L-arginine, L-lysine is capable of significantly preventing heat- and dilution-induced aggregration of lysozyme (Shiraki et al (2002) J Biochem, 132(4):591-5). L-proline has been established as a stabilizer in liquid immunoglobulin preparations like Sandoglobulin® or Privigen®. As an hydrophobic amino acid, it is assumed to interfere with hydrophobic protein-protein interactions and thus protects IgG from denaturation and aggregation. Besides, L-proline exhibits a good safety record when administered to patients with primary immunodeficiencies and was found to represent an amino acid of low toxicity in animal studies (Bolli et al, (2001) Biologicals, 38(1):150-7).
Recent studies contemplate citrate buffer as beneficial in monoclonal antibody formulations as it efficiently minimizes degradations like asparagine deamidations (Zheng and Janis, (2006) Int J Pharm, 308(1-2):46-51). Another advantage of citrate buffer is its capacity to stabilize pH during freezing while the established phosphate buffer system shows the greatest change in pH when lowering temperatures from +25 to −30 degrees C. (Kolhe et al, (2010) Biotechnol Prog, 26(3):727-33).
Fusion proteins may generate a variety of degraded and aggregated products which subsequently may lead to reduced activity and even adverse effects like immunogenicity. Thus, there is still a need for a stable liquid formulation of the fusion protein TNFR:Fc.
Such a formulation shall fulfil a variety of tasks. It has to be physiologically acceptable and preferably provides an environment which guarantees stability of the biopharmaceutical drug in a therapeutically effective concentration. Furthermore, the formulation shall enable a satisfactory shelf-life of the drug.
It is thus the object of the present invention to provide pharmaceutical formulations for TNFR:Fc which can be used as an alternative to those formulations known from prior art. Another object of the present invention is to provide pharmaceutical formulations for TNFR:Fc which are advantageous compared to formulations known from prior art. It is yet another object of the present invention to provide pharmaceutical formulations for TNFR:Fc which cause less drug aggregation than formulations known from prior art.
The present invention demonstrates that, by replacing, e.g., the commonly used phosphate buffer to a citrate buffer system and the stabilizer arginine to lysine, the physical stability of TNFR:Fc can be significantly improved. The proposed buffer citric acid and stabilizer lysine protect TNFR:Fc against degradation induced by mechanical and temperature stress (25 and 40° C.) and at intended storage at 2-8° C. protein degradation was significantly lower in proposed formulations compared to the commonly used phosphate buffered formulations. Therefore, the quality parameters relating to physical stability of the product could be improved. The increased physical stability of the drug product enables a prolonged shelf-life compared to the common product formulations and ensures product safety.